Epimorphic regeneration and related hydrogel delivery systems

ABSTRACT

Methods and compositions are described for enhancing tissue regeneration or wound repair in a mammalian subject comprising a composition comprising (a) a proline hydroxylase inhibitor component or molecule that increases or upregulates HIF1a and (b) a carrier component comprising a hydrogel.

This application claims priority to and the benefit of application Ser.No. 61/962,637 filed Nov. 13, 2013, the entirety of which isincorporated herein by reference.

This invention was made with government support under grant numbers RO1DE021215, DE021104 and P30 CA010815 awarded by the National Institutesof Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Wound repair and regeneration are two separable biological processes bywhich organisms heal wounds. As a model of regeneration in mammals, earhole closure, as seen in rabbits, in the inbred mouse strains MRL/MpJand LG/J and other mutant mouse strains shows multiple similarities tolimb regeneration in amphibians including the replacement of cartilageand the lack of scarring. Other less well known classical regenerativephenotypes are also seen in the MRL ear injury model include rapidre-epithelialization, enhanced tissue remodeling, basement membranebreakdown, blastema growth and re-differentiation not seen during woundrepair. Inflammation is now considered a factor in regenerativeprocesses and its role in ear hole closure has been further demonstratedin genetically selected, pro-inflammatory AIR (acute inflammatoryreactivity) mice which have the ability to close ear holes. Anotheraspect of adult MRL mice is the use of aerobic glycolysis for normalmetabolism, which may contribute to the regenerative response. Thismetabolic state contributes to inflammation, with glycolysis playing animportant role in migration and activity of inflammatory cells. Themolecule hypoxia inducible factor (HIF1a) is a central node in all ofthese and could potentially be a main actor in the MRL ear hole closureresponse.

HIF1a is an oxygen-regulated protein which functions as part of aheterodimeric complex formed with HIF1b in the nucleus and binds to DNAat specific promoter or enhancer sites (i.e., HREs or hypoxia responseelements), thereby regulating the transcription of over 100 geneproducts. These include molecules of interest in regenerative processesinvolved in 1) angiogenesis through the induction of VEGF, VEGFR-1, andPDGF and erythropoietin (EPO); 2) tissue remodeling with the inductionof uPAR, MMP2 and 9 and TIMPs; and 3) glycolytic metabolism with theinduction of lactate dehydrogenase (LDH) which converts pyruvate intolactate and of pyruvate dehydrogenase kinase (PDK) which blockspyruvate's entry into the TCA cycle. HIF1a protein is generallyshort-lived in the cytoplasm because under normoxic conditions, it iscontinually being hydroxylated by prolyl hydroxylases (PHDs), then boundby the von Hippel-Lindau tumor suppressor protein (pVHL) and the morerecently identified SAG/ROC/RBX2 E3 ubiquitin ligase complex whichtargets the molecule for proteolysis. (M. Tan, Q. Gu, H. He, D.Pamarthy, G. L. Semenza, Y. Sun, SAG/ROC2/RBX2 is a HIF-1 target genethat promotes HIF-1 alpha ubiquitination and degradation. Oncogene 27,1404-1411 (2008).) In low oxygen, hydroxylation is inhibited and HIF1aprotein survives and is translocated to the nucleus where it binds HIF1band can now function as a transcription factor, binding to theappropriate DNA elements or HRE. (R. H. Wenger, D. P. Stiehl, G.Camenisch, Integration of oxygen signaling at the consensus HRE. Sci.STKE 2005, re12 (2005).)

The stabilization of HIF1a protein has been accomplished through theinhibition of PHDs, molecules actively involved in collagen secretionand crosslinking PHDs control collagen deposition in fibrosis, responseto ischemia, and wound repair. (See, e.g., X. J. Zhang, L. X. Liu, X. F.Wei, Y. S. Tan, L. Tong, R. Chang, G. Marti, M. Reinblatt, J. W. Harmon,G. L. Semenza, Importance of hypoxia-inducible factor 1 alpha in thehealing of burn wounds in murine model. Wound Repair Regen. 17, A87-A87(2009); T. J. Franklin, W. P. Morris, P. N. Edwards, M. S. Large, R.Stephenson, Inhibition of prolyl 4-hydroxylase in vitro and in vivo bymembers of a novel series of phenanthrolinones. Biochem. J. 353, 333-338(2001); I. Kim, J. E. Mogford, C. Witschi, M. Nafissi, T. A. Mustoe,Inhibition of prolyl 4-hydroxylase reduces scar hypertrophy in a rabbitmodel of cutaneous scarring. Wound Repair Regen. 11, 368-372 (2003).)Considering the impact of scar formation on regeneration, inhibition ofPHDs could have a two-fold effect; the up-regulation of HIF1a and thedown-regulation of scarring. In a chronic diabetic wound model, the useof PHD-inhibiting compounds applied locally to a wound can acceleratewound repair in the presence of increased vascularity and granulationtissue. (I. R. Botusan, V. G. Sunkari, O. Savu, A. I. Catrina, J.Grunler, S. Lindberg, T. Pereira, S. Yla-Herttuala, L. Poellinger, K.Brismar, S. B. Catrina, Stabilization of HIF-1 alpha is critical toimprove wound healing in diabetic mice. Proc. Natl. Acad. Sci. U.S.A.105, 19426-19431 (2008).)

However, the effectiveness of various PHD inhibitor compounds can becompromised by low solubility under physiological conditions,inefficient routes of administration and/or untimely delivery oftherapeutic dose levels. There remains an on-going concern in the art toprovide a delivery system and methodology to better utilize the benefitsand advantages available through such inhibitor compounds.

SUMMARY OF THE INVENTION

In light of the foregoing, it can be an object of the present inventionto provide one or more methods for epimorphic regeneration and/orrelated hydrogel delivery systems, thereby overcoming variousdeficiencies and shortcomings of the prior art, including those outlinedabove.

Other objects, features, benefits and advantages of the presentinvention from this summary and the following descriptions of certainembodiments, and will be readily apparent to those skilled in the arthaving knowledge of various hydrogel drug delivery systems and methodsfor tissue regeneration. Such objects, features, benefits and advantageswill be apparent from the above as taken into conjunction with theaccompanying examples, data, figures and all reasonable inferences to bedrawn therefrom, alone or with consideration of the reference(s)enclosed herein.

In one aspect, a method of tissue regeneration or epimorphicregeneration is disclosed which comprises providing a compositioncomprising (a) a molecule that increases or upregulates HIF1a, such as aproline hydroxylase inhibitor component and (b) a pharmaceuticallyacceptable carrier component, such as a carrier comprising a hydrogel.

Without limitation, in certain embodiments, component (a) of thecomposition being administered can include an inhibitor which is aprodrug of 1,4-DPCA or 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid,or a salt thereof 1,4-DPCA is a selective and potent inhibitor of prolyl4-hydroxylase and has been shown to potently increase cellular HIF1aprotein levels. 1,4-DPCA is registered as CAS 331830-20-7; has amolecular weight of 240.2 and a molecular formula of C₁₃H₈N₂O₃. Thestructure of 1,4 DPCA is

The drug 1, 4-DPCA is publically available from a number of sources,e.g., Cayman Chemical, Enzo Life Sciences, etc.

In another embodiment, such a prodrug can comprise a poly(ethyleneoxide) component. In certain such embodiments, such a component can be apoly(alkylene oxide) block copolymer. In another embodiment, additionalmolecules are anticipated to work in the compositions described hereinin a manner similar to the exemplified 1,4-DPCA. Still other smallmolecules or drugs that increase or upregulate HIF1a are anticipated tobe similarly useful in the compositions and methods described herein.

In another embodiment, the molecule or drug that increases orupregulates HIF1a is DMOG or dimethyloxallyl glycine, or a salt thereof.DMOG is registered as CAS 89464-63-1, has a molecular weight of 175.1and a molecular formula of C₆H₉NO₅. The structure of DMOG is

The drug DMOG is publically available from a number of sources, e.g.,Cayman Chemical, Enzo Life Sciences, etc.

In another embodiment, the molecule or drug that increases orupregulates HIF1a is DFX or desferrioxamine B, also known as30-amino-3,14,25-trihydroxy-3,9,14,20,25-pentaazatriacontane-2,10,13,21,24-pentone, or a salt thereof. DFX isregistered as CAS 70-51-9; has the formula C₂₅H₄₈N₆O₈ and a molecularweight of 560.68. The drug DFX is publically available from a number ofsources, e.g., Sigma Chemical.

In another embodiment, the molecule or drug that increases orupregulates HIF1a is1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine or Imiquimod, or asalt thereof. Imiquimod has a molecular formula of C₁₄H₁₆N₄ and amolecular weight of 240.3. Its structural formula is:

The drug Imiquimod is publically available from a number of sources.

In another embodiment, the molecule or drug that increases orupregulates HIF1a is cobalt II chloride or CoCl₂. CoCl₂ is availablefrom a number of sources, e.g., Sigma-Aldrich. These additionalmolecules are anticipated to work in the compositions described hereinin a manner similar to the exemplified 1,4-DPCA. Still other smallmolecules or drugs that increase or upregulate HIF1a are anticipated tobe similarly useful in the compositions and methods described herein.

In another aspect, a method for enhancing tissue regeneration or woundrepair in a mammalian subject, comprises administering to the subject inneed thereof a composition comprising a molecule that up-regulates HIF1ain a pharmaceutically acceptable carrier. In one embodiment, thecomposition comprises (a) a proline hydroxylase inhibitor component ormolecule that increases or upregulates HIF1a; and (b) a carriercomponent comprising a hydrogel. In another embodiment, the compositionemploys as the upregulating HIF1a molecule 1, 4-DPCA, DMOG, DFX,Imiquimod or CoCl₂. In certain embodiments, the administration involvescontacting a cellular medium with the composition. In anotherembodiment, the method comprises systemically administering to a subjectin need thereof the composition described herein. In another embodiment,the method comprises administering the composition to a mammaliansubject in need thereof at a site distal from the site of a wound orinjury. In another embodiment, the method comprises administering thecomposition to a mammalian subject in need thereof at a site local oradjacent to the site of a wound or injury.

In another embodiment, the method further comprises administering thecomposition at a drug release rate which achieves maximal HIF1aupregulation in vitro. In another embodiment, the method involvesadministering the composition and releasing the HIF1a up-regulator at acontinuous rate over at least 4 days.

In part, the present invention can be directed to a method of epimorphicregeneration. Such a method can comprise providing a compositioncomprising a proline hydroxylase inhibitor component and a carriercomponent comprising a hydrogel comprising a condensation product of

wherein R₁ and R₂ can be independently selected from polyhydric coremoieties; X can be selected from divalent linker moieties; n1 and n2 canbe integers from 1 to about 201; p1 and p2 can be integers independentlyselected from 2 to about 10, corresponding to the polyol from which R₁and R₂ are derived; NHS can be a N-hydroxysuccinimido moiety; and Cyscan be an N-terminal cysteine residue; and contacting such a prolinehydroxylase inhibitor component with a cellular medium expressing aproline hydroxylase, such a component as can be in an amount over a timeat least partially sufficient to produce a regenerative response.Without limitation, such a regenerative response can be considered inthe context of a functional effect achieved therewith, such a functionaleffect including but not limited to enhanced tissue remodeling response,de-differentiated cellular signature, increased glycolytic enzymes,increased components of inflammatory response and increasedangiogenesis. In certain embodiments, such a cellular medium can bewithin a non-regenerative mammal presenting a tissue injury. In certainsuch embodiments, such a composition can be administered distal to suchan injury. Without limitation, administration can comprise subcutaneousinjection and, with respect to certain such embodiments, multipleinjections over time.

Regardless, with respect to certain embodiments, R₁ and R₂ can beindependently selected from hexaglycolic and tripentaerythritolicmoieties, such that each of p1 and p2 can be 8. In certain suchembodiments, X can be a C₄-C₆ dicarbonyl moiety. In certain suchembodiments, X can be selected from C(O)(CH₂)₃(CO), C(O)CH₂OCH₂C(O),C(O)CH₂CH(CH₃)CH₂C(O), C(O)(CH₂)₂C(O) and C(O)CH₂CH(CH₃)C(O) moieties.Without limitation, in certain embodiments, such an inhibitor componentcan comprise a prodrug of 1,4-DPCA. In certain such embodiments, such aprodrug can comprise a poly(ethylene oxide) component. In certain suchembodiments, such a component can be a poly(alkylene oxide) blockcopolymer.

In one embodiment, a carrier is a hydrogel delivery system comprising aderivatized polyethylene glycol (PEG) that crosslinks in situ viaoxoester mediated native chemical ligation. One such hydrogel deliverysystem comprises a derivatized polyethylene glycol (PEG) that crosslinksin situ via oxoester mediated native chemical ligation. The hydrogel cancomprise a derivatized PEG, such as PEG-Cys8 (P8Cys) or PEG-NHS8(P8NHS), and be prepared as described in detail in Strehin et al, 2013,Biomater. Sci., 2013, 1, 603-613 incorporated by reference herein.

In yet further embodiments, the term “pharmaceutically acceptablecarrier” can include a biologically compatible fluid medium, solution orpharmaceutically acceptable delivery vehicle suitable for the form ofthe composition. The various components of the composition may beprepared for administration by being suspended, dispersed or dissolvedin a pharmaceutically or physiologically acceptable carrier such asisotonic saline; isotonic salts solution or other formulations that willbe apparent to those skilled in such administration. Other aqueous andnon-aqueous isotonic sterile injection solutions and aqueous andnon-aqueous sterile suspensions known to be pharmaceutically acceptablecarriers and well known to those of skill in the art may be employed forthis purpose. As used herein the term “pharmaceutically acceptablecarrier” or “diluent” is intended to further include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with administration to humans. In one embodiment, the diluentis saline or buffered saline.

Still other examples of carrier materials suitable for systemic, distal,or local administration of the composition comprising theHIF1a-increasing or up-regulating composition may be selected by one ofskill in the art, such as biomaterials admixed with the HIF-1a inducingcomposition, which are able to be implanted in the site of the wound. Anexample of such a material are those described in, e.g., Ni, et al, 2011Internat. J Nanomedicine, 6:3065-3075.

In one embodiment, the use of a hydrogel system to deliver 1,4-DPCApermits the drug to be released at a rate that achieves a reliable andpredictable steady state of drug concentration in vivo. This controlledrelease of a composition that achieves a maximal HIF1a upregulation invitro increases HIF1a, while decreasing the toxicity or side effectsthat may be observed in upon other modes of delivery of the molecule.Regenerative effects of the use of certain embodiments of thesecompositions are described in detail below and in the examples.

The method of tissue regeneration or epimorphic regeneration alsocomprises contacting such a composition containing component (a) and (b)with, or administering the composition into, a cellular medium. In oneembodiment, the medium expresses a proline hydroxylase.

By “cellular medium” as used herein is meant an ex vivo samplecontaining mammalian cells or an in vivo tissue or mammalian cells,e.g., in a mammalian subject. In certain embodiments, such a cellularmedium containing mammalian cells can be an in vitro sample containingcells, such as a sample of cells removed from a mammalian subject,cultured ex vivo, and contacted ex vivo with the compositions asdescribed herein. Such an ex vivo-contacted cellular medium is thenitself transferred or reimplanted to the site of cellular or tissueinjury in the mammalian subject. In another embodiment, the cellularmediam refer to cells or tissue within a mammal presenting a tissueinjury, so that the contacting step involves in vivo administration tothe mammalian subject of the composition described herein. A mammaliansubject includes a human, a veterinary or farm animal, a domestic animalor pet, rodents and animals normally used as mammalian models forclinical research, such as the mouse models described in the examples.

For use in the contacting step, the proline hydroxylase inhibitor/HIF1aincreasing drug component is present in the composition in an amountthat is at least partially sufficient to produce a regenerative responsein the cells of the cellular medium. The contacting step can occur for atime at least partially sufficient to produce a regenerative response.Without limitation, a regenerative response can be defined as thefunctional effect achieved by contact between the cells of the sampleand the composition. Such a functional effect includes but is notlimited to enhanced tissue remodeling response, de-differentiatedcellular signature, increased glycolytic enzymes, increased componentsof inflammatory response and increased angiogenesis.

In one embodiment, the methods of this invention comprise systemicallyadministering the composition to a subject in need thereof. In anotherembodiment, the methods described herein involve administering to asubject in need thereof the composition at a site distal to the cells ortissue to be regenerated, e.g., distal to the wound. In anotherembodiment, the methods described herein involve locally administeringat or near the site of the wound in a subject in need thereof thecomposition in an amount that permits sufficient HIF1a upregulation toinduce wound healing or tissue repair with minimal toxicity to thesubject. In one embodiment, the composition is administered at a drugrelease rate which achieves maximal HIF1a upregulation when assayed invitro. In one embodiment, the concentration of the HIF1a-upregulatingmolecule is kept at a concentration low enough to avoid toxic sideeffects.

In another embodiment, the composition releases the HIF1a up-regulatorat a continuous, non-toxic rate over at least 1 or more days, at leastone week, at least two weeks, or more than 3 weeks. In certainembodiments, the composition is administered for at least one week, orat least 4 weeks at a desired concentration level to enhance the pace ofwound healing. The level of administration may be selected or adjustedbased upon the nature and site of the wound or tissue being regenerated.

The method may be accomplished by administering the appropriateconstruct or composition by a suitable route. In one embodiment of themethods, the compositions are administered directly into the subject orinto the subject's tissue requiring repair or regeneration, wherepossible. Conventional and pharmaceutically acceptable routes ofadministration include, but are not limited to, systemic routes, such asoral intake and subcutaneous administration. In one embodiment, such acomposition can be administered distal to such an injury. Withoutlimitation, administration can comprise subcutaneous injection. Otherroutes of administration may include intraperitoneal, intravenous,intranasal, intravenous, intramuscular, intratracheal, topical, andother parenteral routes of administration. Routes of administration maybe combined, if desired. In some embodiments, the administration isrepeated periodically by multiple doses and/or injections over time.

Dosages of these therapeutic compositions employed in these methods willdepend primarily on factors such as the tissue or injury being treated,the age, weight and health of the patient, and may thus vary amongpatients. Methods for determining the timing of frequency ofadministration or use of continuous release will include an assessmentof tissue response.

In another embodiment, the method further comprises administering to thesubject along with the therapeutic compositions that increase orup-regulate HIF1a, an adjunctive therapy directed toward the tissuebeing treated.

In another aspect, a method of tissue regeneration or epimorphicregeneration is disclosed which comprises providing a compositioncomprising (a) a molecule or drug that increases or upregulates HIF1aand (b) a carrier component comprising a hydrogel as described above.

In one aspect, the proline hydroxylase inhibitor molecule or drug thatincreases or upregulates HIF1a is 1, 4-DPCA or1,4-dihydrophenonthrolin-4-one-3-carboxylic acid, or a salt thereof1,4-DPCA is a selective and potent inhibitor of prolyl 4-hydroxylase andhas been shown by others to potently increase cellular HIF1a proteinlevels. 1,4-DPCA is registered as CAS 331830-20-7; has a molecularweight of 240.2 and a molecular formula of C₁₃H₈N₂O₃. The structure of1,4 DPCA is

The drug 1, 4-DPCA is publically available from a number of sources,e.g., Cayman Chemical, Enzo Life Sciences, etc.

In another embodiment, the molecule or drug that increases orupregulates HIF1a is DMOG or dimethyloxallyl glycine, or a salt thereof.DMOG is registered as CAS 89464-63-1 and has a molecular weight of 175.1and a molecular formula of C₆H₉NO₅. The structure of DMOG is

The drug DMOG is publically available from a number of sources, e.g.,Cayman Chemical, Enzo Life Sciences, etc.

In another embodiment, the molecule or drug that increases orupregulates HIF1a is DFX or desferrioxamine B, also known as30-amino-3,14,25-trihydroxy-3,9,14,20,25-pentaazatriacontane-2,10,13,21,24-pentone, or a salt thereof. DFX isregistered as CAS 70-51-9; has the formula C₂₅H₄₈N₆O₈ and a molecularweight of 560.68. The drug DFX is publically available from a number ofsources, e.g., Sigma Chemical.

In another embodiment, the molecule or drug that increases orupregulates HIF1a is1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine or Imiquimod, or asalt thereof. Imiquimod has a molecular formula of C₁₄H₁₆N₄ and amolecular weight of 240.3. Its structural formula is:

The drug Imiquimod is publically available from a number of sources.

In another embodiment, the molecule or drug that increases orupregulates HIF1a is cobalt II chloride or CoCl₂. CoCl₂ is availablefrom a number of sources, e.g., Sigma-Aldrich.

Without limitation, in certain embodiments, such an inhibitor componentcan comprise a prodrug of 1,4-DPCA. In certain such embodiments, such aprodrug can comprise a poly(ethylene oxide) component. In certain suchembodiments, such a component can be a poly(alkylene oxide) blockcopolymer.

In part, the present invention can also be directed toward a method ofmodulating proline hydroxylase activity. Such a method can compriseproviding a composition comprising a prodrug of 1,4-DPCA and a carriercomponent comprising a hydrogel comprising a chemical ligation productof

wherein R₁ and R₂ can be independently selected from polyhydric coremoieties of the sort described herein; X₁ can be selected from divalent(CH₂)₃, CH₂OCH₂, CH₂CH(CH₃)CH₂, (CH₂)₂, and CH₂CH(CH₃) moieties; and n1,n2, p1 and p2 can be as described elsewhere herein; and contacting sucha 1,4-DPCA component of such a prodrug with a cellular medium expressinga proline hydroxylase, such a component as can be in an amount at leastpartially sufficient to modulate, inhibit or otherwise affect prolinehydroxylase activity in such a cellular medium. Without limitation, sucha cellular medium can be within a non-regenerative mammal presenting atissue injury.

In certain embodiments, such administration can be distal to an injury.Without limitation, administration can be by subcutaneous,intraperitoneal and/or intramuscular injection and, with respect tocertain such embodiments, can comprise multiple injections over time tostabilize a constitutive cellular level of HIF1a protein. Regardless, incertain embodiments, each of R₁ and R₂ can be a hexaglycolic moiety,such that each of p1 and p2 can be 8, and X can be a (CH₂)₃ moiety.Without limitation, 1,4-DPCA can be coupled to a poly(alkylene oxide)block copolymer.

In part, the present invention can be directed to a drug deliverysystem. Such a system can comprise a first macromonomer componentcomprising a first reactive moiety; a second macromonomer componentcomprising a second reactive moiety reactive with such a first reactivemoiety; and a drug component selected from proline hydroxylase inhibitorcompounds and prodrugs thereof. Without limitation, such a prodrug cancomprise such a proline hydroxylase inhibitor compound coupled to acleavable polymer component. In certain embodiments, each suchmacromonomer component can be in a fluid medium. In certain otherembodiments, upon contact one with the other, such first and secondmacromonomer components can be cross-linked to provide a hydrogel, andsuch a drug component can be suspended or dispersed therein. Regardless,such a drug component can be selected from 1,4-DPCA, a poly(alkyleneoxide) coupled prodrug of 1,4-DPCA, DMOG, DFX, Imiquimod and CoCl₂ orother proline hydroxylase inhibitor compounds as would be understood bythose skilled in the art. Likewise, such a drug delivery system would beunderstood by those skilled in the art made aware of this invention,such delivery systems, comprising first and second macromonomercomponents, as are described in co-pending application Ser. No.13/798,744 filed Mar. 13, 2013, incorporated herein by reference in itsentirety and as discussed more fully below. Accordingly, with respect tosuch a drug delivery system, a first reactive moiety of such a firstmacromonomer component can be an N-hydroxysuccinimide ester moiety, anda second reactive moiety of such a second macromonomer component can bean N-terminal cysteine moiety.

In part, the present invention can also be directed toward a method ofusing a hydrogel system to modulate cellular levels of HIF1a protein.Such a method can comprise providing a first hydrogel precursorcomponent comprising a fluid and/or aqueous medium comprising

a second hydrogel precursor component comprising an aqueous mediumcomprising

wherein R₁, R₂, n1 and n2 can be as described above, and a prolinehydroxylase inhibitor precursor component comprising 1,4-DPCA coupled toa poly(alkylene oxide) block copolymer; mixing such precursor componentsto provide a hydrogel comprising such a coupled 1,4-DPCA componenttherein; and administrating such a hydrogel to a non-regenerative mammalpresenting a tissue injury. Such administration can be distal to such aninjury to provide 1,4-DPCA in an amount as can be sufficient toup-regulate HIF1a protein at the site of such an injury. Withoutlimitation, in certain embodiments, administration can comprisesubcutaneous, intraperitoneal and/or intramuscular injection. In certainsuch embodiments, administration can comprise multiple injections overtime to stabilize a constitutive cellular level of HIF1a protein.Regardless, in certain embodiments of the sort described herein, aportion of such a proline hydroxylase inhibitor precursor component canbe introduced to each of such first and second hydrogel precursorcomponents prior to mixing.

In part, the present invention can also be directed to a therapeuticcompound comprising, for instance, at least one 1,4-DPCA moiety coupledto via a cleavable (e.g., without limitation, hydrolyzable) moiety to apolymer component such as, for instance, a poly(alkylene oxide)component. Without limitation, in certain embodiments, such a polymericcomponent can comprise at least one ethylene oxide monomeric unit, atleast one propylene oxide monomeric unit or a combination of suchmonomeric units. In certain such embodiments, such a component cancomprise poly(ethylene oxide) or a copolymer of poly(ethylene oxide) andpoly(propylene oxide). Without limitation, such a compound can comprisesuch a copolymer coupled at each terminus to a 1,4-DPCA moiety. Inaccordance with certain aspects of this invention, reference is made tothe drug conjugate of Example 11, below. In certain other embodiments,such a compound can comprise a plurality of poly(ethylene oxide) and/orpoly(propylene) oxide components coupled to a polyhydric core moiety.Without limitation, such a polyhydric core can be selected fromhexaglycolic and tripentaerythritolic moieties. Regardless, one or moresuch poly(alkylene oxide) components can be further coupled to a1,4-DPCA moiety. In accordance with certain aspects of this invention,reference is made to the drug conjugate of Example 18, below.

BRIEF DESCRIPTION OF THE DRAWINGS

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FIGS. 1A, B, Ca-b, D, Ea-d, F, G and Ha-c. HIF1a levels areover-expressed and required in the MRL regenerative response. (A)Diagram of ear punch injury, derived histological sections and processedtissue samples is shown. (B-F) show HIF1a protein expression levels inMRL pre- and post-injury compared to B6 mice. (B) Pooled ear hole donuts(n=4) were processed for HIF1a western blot (WB) analysis using comassieblue (red on Odyssey) as loading control; N=2. (Ca-b) Ear tissue wasused for HIF1a immunostaining and photomicrographs. Scale bar=0.1 mm.(D) Multiple samples for each timepoint (n=3-5; N=2) were quantitativelyanalyzed with highly significant differences p<0.01 (**), at alltimepoints between B6 and MRL responses peaking on da7. (Ea-d) Furtherconfirmation of HIF1a levels was carried out using MRL and B6 micebackcrossed (BC4) to transgenic HIF1a-peptide-luciferase reporter mice.MRL.HIF-luc and B6.HIF-luc mice showed levels of luciferase activitywith (F) IVIS-detected photon number on da7 in healing ear and wholemouse in MRL (red) compared to the B6 cross (blue), expressed as(p/sec/cm2/sr)=photons/sec/cm2/steradian. Both E and F arerepresentative of experiments (N=3) of n=5 mice/group. (G-Ha-c) showrequirement for HIF1a in MRL ear-hole closure regenerative responseswith in-vivo RNAi against Hif1a (siHif1a) treatment. (G) SiHIF1a's(Qiagen) tested for blocking Hif1a mRNA in-vitro in MRL ear cells (nreplicates=3); Gapdh is control (N=2) (H) SiHif1a_3 was used in-vivo inMRL.HIF-luc mice treated, ear-punched, and followed for 28 days. Micewere either injected subcutaneously with in-vivo-JETPEI-siHif1a_3mixture da0-20 (15 ug/mouse; green) or PBS (red) with ear hole closure(Ha) highly significantly blocked, on da28 p=0.000016 (**); (n=4mice/group; N=2). (Hb) HIF1a levels determined by bioluminescence(dorsal and ventral) in reporter mice under treatment with siHif1a_3.(Hb,c) Number of photons detected on da14 in the injured ear (p=0.03) orwhole mouse (p=0.04) with significant differences (*), by siHif1a_3compared to control (n=4 samples/group; N=2).

FIGS. 2A, B, C, Da-c and E. (A) Diagram showing that 1,4-DPCA acts as aninhibitor to PHDs and slows down degradation of HIF-1α. (B) Chemicalstructures of the drug delivery components including 1,4-DPCA, PluronicF127, P8NHS, and P8Cys. (C) The presence of drug microcrystals does notinterfere with the gelation kinetics of the hydrogel. (Da-c) The drugwas encapsulated in the hydrogels to yield white and opaque cylindricalhydrogels (left) which when incubated in PBS released the drug leavingbehind clear hydrogels (right). (E) In-vitro experiments showed thatdrug release occurred over several days for hydrogels containing 119-477ug drug. Dashed lines represent total drug loading for each formulation.C and E show replicate samples (n=3).

FIGS. 3Aa-c, B, C, D, E, Fa-f and G. 1,4-DPCA drug/gel stabilizes HIF1a,but not HIF2a, in-vitro and in-vivo. (Aa-c) B6 ear fibroblast-like cellswere cultured with normal medium (Nor), gel alone (G₀) or 2 mg/mldrug/gel (G_(d)) using 100 ul total volume formed as a solid disc in 24well plates. Addition of 1,4-DPCA drug/gel induced HIF1a protein,determined by immunostaining (G_(d), green, Ac). Scale bar=50 um. (B)Cell lysates (n=3/lane) from (A) were used for western blot (WB)analysis for HIF1a (green) and HIF2a (red) levels compared to controlprotein a-tubulin (red) (N=4). (C) For activation of HIF1a target genetranscription, RT-PCR analysis of treated-B6 cell mRNA (n=3) from (A)examined multiple genes increased by 1,4 DPCA/gel includingpro-angiogenic target genes Vegf and Hmox1, and pro-glycolytic targetsLdh-a, Pgk, Pdk1, and Glut1. Gapdh and 18S rRNA were used as internalcontrols for all RT-PCR reactions (N=4). (D-G) Mice treated with singleinjections of drug/gel and tested for HIF1a upregulation. (D) Schematicillustrates in-vivo treatment schedule. SW mice were ear-punched andinjected several hours later in back of neck with either G₀ or G_(d).Ear donut tissue for protein and IHC was collected on da1-5. In (E),hole donuts (n=6) were processed and western blot (WB) analysis wascarried out with antibody to HIF1a (green) and analyzed (Odyssey) (N=3).The loading control is comassie-stained sample appearing red on theOdyssey. In (Fa-f), immunostaining of ear tissue with anti-HIF1a (green)and DAPI (blue) was carried out. (G) IHC quantitation for G₀ (blue) andG_(d) (red) tissue show highly statistically significant differences,p<0.01 (**), at all timepoints (with mean+/−SE shown for all samples,n=5/treatment group, N=2).

FIGS. 4A, Ba-d, Ca-d, D and E. Sequential injections of 1,4-DPCAdrug/gel into mice at 5 day intervals shows a significant effect onhealing. (A) Injection scheme diagram shows SW mice ear-punched andinjected on da0, 5, and 10 into separate locations at base of neck, i.e.one site every five days (arrow). (Ba-d) Mice (n=16/group; N=4) wereinjected with either G₀ (0 mg/ml drug/gel, blue line) or G_(d) (2 mg/mldrug/gel, red line) and followed for 35 days. Healing results with threeinjections of 2 mg/ml drug/gel are compared to G₀ on da35, p=1.93E-08(**). On the left are representative da35 ear pinnae (arrows point earholes) (Ba, 0 mg drug/gel; Bb-c, 2 mg drug/gel). (Ca-d) Histologicalexamples of Alcian blue-stained, da35 1,4-DPCA-treated closed ear holes(Ca,b) with the 2 mm area of original hole indicated and seen at highermagnification (Cc,d) (n=2). Areas of condensation are shown (blackarrows); blue staining indicates presence of proteoglycans. (Cc) showsan earlier time-point after closure where epithelial cells (red arrow)persist in the new bridge region; in (Cd), the bridge is filled withmesenchymal cells. (D) Hole donuts (n=6) from punched ears treated withG₀ or G_(d) were processed and western blot (WB) analysis (Odyssey) wascarried out (N=3) with anti-HIF1a (green) or anti-HIF2a (red)antibodies; comassie protein staining is loading control (red). (E) Onda20, in-vivo siHif1a treatment showed highly significant inhibition,p=7.79E-06 (**) comparing 1,4-DPCA-induced (G_(d))/G₀ ear hole closure(red/green lines). siHif1a treatment compared to G₀-treated SW ears(green/blue) with significant, p=0.05 (*) inhibition. Highly significantp=0.01 (**) differences are seen comparing G₀ to G_(d) (red/blue)(n=6-8; N=2).

FIGS. 5A-C. HIF1a stabilization by 1,4-DPCA drug/gel induces stem cellmarkers in-vitro. (A) Cultured MRL (upper panels) and B6 (middle panels)cells grown on coverslips were immunostained with anti-HIF1, NANOG,OCT3/4, CD133, PAX7, PREF1 (DLK1), NESTIN, vWF and CD34. B6 cells werecultured with 2 mg/ml drug/gel (B6+G_(d), lower panels) or 0 mg/mldrug/gel (G₀ not shown) for 24 hours (n=5-10 fields/coverslip; N=3). (B)QPCR results, consistent with IHC, show highly significant changes,p<0.01 (**) in mRNA levels for all molecules in drug-treated cellpopulations (n=3; N=3). (C) MRL cells (n=3 coverslips/grp) were treatedwith either siRNA control (left panel) or siHif1a (right panel) for 48hours. The cells were immunostained with anti-NANOG antibody (N=2). Allscale bars=50 um.

FIGS. 6Aa-i, Ba-g, Ca-e, Da-e and Ea-k. Key Elements of Regeneration.(Aa-i) Re-epithelialization of H&E-stained punched ear tissue (n=4/grp)is seen on da2 after injury from G₀ or G_(d)-treated SW mice (Aa,b) withblack arrows showing incomplete (Aa) vs complete epidermis (Ab).Immunostaining is seen for HIF1a (Ac,d) and WNT5a (Ae,f) for G₀ (Ac,e)and G_(d) (Ad,f) treated tissue; white arrows show epidermal staining.IHC of WNT5a protein expression induced in-vitro without (Ag) or with(Ah) 1,4-DPCA drug/gel; (Ai) shows western analysis of pooled tissue(n=3; N=2). (Ba-g) H&E sections (d4) illustrate the area ofG_(d)-treated wound site de-differentiation (Ba,b, dashed lines).Immunostaining in G_(d) and G₀ (insets) tissue include NESTIN, OCT3/4,NF, and PAX7 (Bc-f) (n=4), and qPCR results (Bg) show expression levels(n=3; N=3). (Ca,b) Tissue remodeling with laminin immunostaining todetect basement membrane (white arrow, inset) in G_(d) vs G₀-treatedtissue and (Cc-e) levels of MMP9, MPO, and Ly6G protein in G_(d) vs G₀(insets) are seen (n=4; N=3). In (A-C), blue is DAPI; red or green showsspecific immunostaining (Da-c) PSR staining shows dal4 collagencross-linking in (Da) G₀, (Db) G_(d), (Dc) G_(d)+siHif1a-treated eartissue with (Dd) quantitated polarized light results showing significantdifferences between G₀ and G_(d)-treated and G_(d) andG_(d)+siHIf1a-treated mice. (De) qPCR results show early (da2-3) Loxl4and Ctgf expression in G_(d) and G₀ samples (n=6/grp; N=2). (E) Blastemagrowth and re-differentiation into cartilage and hair follicles is seenafter G_(d) treatment. (Ea) Low magnification histological image of earhole tissue stained with Alcian blue shows differences inchondrogenesis. Solid vertical lines indicate ends of cartilage, brokenlines show soft tissue borders. (Eb) shows quantitation of soft tissueear hole diameter, da35 with (Ec) a larger image of G_(d)-treated tissueshowing two areas of new chondrogenesis (Eg,h) and new hair follicles(Ek). QPCR results show up-regulated chondrogenesis-(Ed) and hairfollicle-associated (Ei) genes on da21 in G_(d) vs G₀-treated tissue(n=3; N=3). Differences in (Ee) cartilage hole diameter and (Ef)cartilage area (a histomorphometric measurement of Alcian blue stainingin new growth area) show highly significant differences. (Ej) The numberof KRT14+ hair follicles in G_(d)-injected mice is compared to normalear tissue or tissue from G_(o)-injected mice. All scale bars=0.1 mmexcept Ah=50 um. Statistical differences are indicated by (*) forsignificant with p<0.05 and (**) for highly significant with p<0.01.

FIG. 7. Synthesis of 1,4-DPCA was accomplished in three steps. Thesynthetic scheme and the chemical structures of the three main products(1-3) are shown here.

FIGS. 8A-B. (A) Synthetic scheme for preparing PEG DPCA or P(TP)8DPCA20K. (B) The amount of 1,4 DPCA remaining bound to the PEG polymer overtime when incubated in pH 7.4 buffer at 37° C.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Illustrating certain non-limiting embodiments of this invention,1,4-DPCA (1,4-dihydrophenonthrolin-4-one-3-carboxylic acid), awell-defined PHD inhibitor, was used to stabilize the constitutive levelof HIF1a protein. A locally-deliverable novel drug hydrogel constructwas designed to slowly deliver 1,4-DPCA from the hydrogel over 4-10 daysin-vitro. A functional measurement of in-vivo drug release, increase andstabilization of the constitutive level of HIF1a was observed over fivedays upon subcutaneous injection of 1,4-DPCA hydrogel. Multipleperipheral subcutaneous injections of 1,4-DPCA hydrogel over a 10-dayperiod led to a regenerative response in non-regenerative Swiss Webstermice in a manner which fully emulates the MRL healing response (e.g., apro-regenerative state with enhanced re-epitheliazation of the wound,induced progenitor cell phenotypes and enhanced tissue remodeling withincreased MMP levels and a resultant basement membrane breakdown). (Bothresponses are inhibited by siHif.) The results demonstrate thatcontrollable regeneration can be achieved by specifically manipulatingthe levels of HIF-1a protein using a peripherally deliverable drug/gelconstruct.

HIF1a Levels are Over-Expressed in the MRL Regenerative Response

To determine HIF1a protein expression levels in MRL vs B6 mice, woundedear pinnae (FIG. 1A) were examined by IHC and western analysis (FIG.1B-D). Strikingly higher HIF1a levels were seen in MRL tissuepost-injury with peak levels on day 7. For longitudinal studies, Hif1areporter mice were created by backcrossing these two strains to thetransgenic HIF1a-peptide-luciferase reporter mouseFVB.129S6-Gt(ROSA)26S, made by fusing luciferase to the domain of HIF1athat binds to pVHL in an oxygen-dependent way (ODD peptide). As seen inFIG. 1E-F, MRL.HIF-luc mice showed high levels of luciferase activity, acorrelate of HIF1a protein levels, compared to the B6.HIF-luc mouse bothpre-injury in the liver and post-injury throughout the body includingthe ear. Inhibition of HIF1a blocks MRL ear hole closure.

To examine if HIF1a is necessary for ear hole closure, Hif1a siRNA wasused. A panel of siHf1a's showed that 1 out of 4 tested siRNAs(siHif1a_3) could completely inhibit constitutive Hif1a mRNA levels inMRL fibroblasts, as well as fibroblasts from B6 and SW mice (FIG. 1G).SiHif1a_3 was then tested in-vivo in MRL.HIF-luc mice for its effect onear hole closure and HIF1a levels. (Previous studies determined that a30-day hole size of 0-0.4 mm diameter represented an MRL regenerativeresponse, and 1.2-1.6 mm diameter hole represented a B6 wound repair andnot a regenerative response.) As seen in FIG. 1H, ear hole closure wasblocked by siHif1a_3 (FIG. 1Ha) and HIF1a levels as determined bybioluminescence were reduced in the reporter mouse whole bodymeasurements as well as in the ear (FIG. 1Hb-c). From these results, thenecessity of increased HIF1a levels for a regenerative response is clearand suggests the possibility that stabilization of HIF1a innon-regenerative mice may lead to a regenerative ear hole closureresponse.

Formulation of a Drug-Loaded Hydro Gel Construct that Stabilizes HIF1aIn-Vitro

1,4-DPCA has been reported to be a potent inhibitor of the hydroxylases(PHDs and FIH) in-vitro and in-vivo at the protein level and rats giventhis compound showed inhibition of collagen hydroxylation and areduction in collagen deposition. (See, Franklin et al, supra.) 1,4-DPCAcan also stabilize HIF1a (FIG. 2A). As a delivery system for 1,4-DPCA, apolymer hydrogel system was devised. Composed of a crosslinked networkof multi-armed polyethylene glycol, it is capable of rapid in-situ gelformation from a liquid precursor. This hydrogel system was chosen asthe delivery vehicle due to its rapid gelation under physiologicalconditions, biocompatibility, and other favorable properties for in-vivouse. Drug-loaded hydrogels were formed by suspending polymer-stabilized1,4-DPCA microcrystals in an aqueous mixture of hydrogel precursorsP8Cys and P8NHS (FIG. 2B), which solidified rapidly upon mixing of bothprecursors to entrap the drug microcrystals within the hydrogel (FIG.2C). In-vitro drug release studies demonstrated the slow and controlleddelivery of 1,4-DPCA from the hydrogel over several days (FIG. 2D-E).Due to the poor solubility of 1,4-DPCA, crystals were surface-modifiedwith F127 polymer to aid in homogeneous distribution throughout thehydrogel. Furthermore, entrapment of drug within the hydrogel helped toavoid cytotoxicity observed upon direct contact between cells and drugcrystals. Due to the low solubility of 1,4-DPCA in water, drug releaseis likely by surface erosion from the crystals and diffusion through thehydrogel matrix.

B6 fibroblasts cultured with this drug/gel combination for 24 hrs.showed increased levels of HIF1a protein, but not HIF2a protein, both inthe cytoplasm as well as in the nucleus as determined by IHC (FIG. 3A)and Western analysis (FIG. 3B) when compared to cells incubated witheither gel alone or no drug/no gel. To determine the activation of HIFtarget gene transcription, RT-PCR analysis of mRNA from treated B6 cellsrevealed that multiple genes were specifically increased by 1,4-DPCAincluding the pro-angiogenic target genes Vegf and Hmox1 and thepro-glycolytic targets Ldh-a, Pgk, Pdk1, and Glut1 (FIG. 3C).Furthermore, all of these genes were blocked by siHif1a_3.

The Effect of the 1,4-DPCA Drug/Gel Construct In-Vivo

To test the drug/gel construct for in-vivo function, non-regenerative SWmice were used. An initial attempt to directly apply the gel to the earhole injury site failed as it could not be maintained on the wound. Thedrug/gel was injected subcutaneously at the base of the neck, distalfrom the wound, to achieve a pharmacological effect.

The kinetics of the effectiveness of the drug/gel construct in theinjured ear was examined to determine how often re-injection would benecessary. After earpunching, a single injection (100 ul) of hydrogelcontaining either 2 mg/ml drug microcrystals (G_(d)) or 0 mg/ml drugmicrocrystals (G₀) was given plus an un-injected group. Ears wereharvested daily for 5 days (FIG. 3D). The effect of 1,4-DPCA in-vivo onHIF1a expression levels in the ear, as determined by Western analysis(FIG. 3E) and by IHC (FIG. 3F,G), showed that HIF1a levels rosebeginning on day 1, peaking on days 3-4 post-injection.

Multiple Injections of the Drug/Gel Construct Leads to a RegenerativeResponse

Given data showing that day 7 post-injury is (approximately) the peak ofHIF1a protein expression in MRL mice during healing and that HIF1aprotein is still elevated in MRL on day 14 (FIG. 1D), multipleinjections of drug/gel were given. Since a single injection of drug/gelhad an effect on HIF1a levels through day 5, drug/gel and control gelwere injected subcutaneously every 5 days (day 0, 5 and 10) into threeadjacent sites in the back of the neck (FIG. 4A). In FIG. 4B, highlysignificant (on day 35 p=1.93E-08) as well as complete ear hole closure(FIG. 4C) was achieved via multiple injections of 2 mg/ml drug/gel(G_(d)) compared to 0 mg/ml drug/gel alone (G₀) with ongoing ear holeclosure occurring up to day 35. Furthermore, western analysis (FIG. 4D)showed that although HIF1a is slightly up in G₀-treated SW mice on day 7post-injury, it is dramatically up in G_(d)-treated SW on day 7, similarto MRL (FIG. 1B), and is still over-expressed up to day 21. On thecontrary, HIF2a was barely affected by 1,4-DPCA in-vivo, consistent within-vitro experiments using other cell types and showing no effect of1,4-DPCA on HIF2a expression.

It should be noted that the injection of drug/gel at sites on the backof the neck to achieve ear hole regeneration suggests a systemic ratherthan a local/topical drug effect—a promising outcome since systemicactivity may allow regeneration in less accessible anatomic sites and ina variety of tissues.

Distal Effects of the 1,4-DPCA Drug/Gel Construct

Mice were injected subcutaneously (day 0, 5 and 10) into the left andright flank regions with drug/gel. Partial ear hole closure was achievedat 2 mg/ml but not 1 mg/ml. Though clearly effective, injection with 2mg/ml drug/gel at the more distal flank sites led to lesser closure. Wealso examined long-term effects of gel with or without drug and nohistopathological effects or weight changes were seen at 3 months.

SiHif Blocks the Regenerative Effect of 1,4-DPCA Drug/Gel In-Vivo

Finally, SW mice injected 3 times with 2 mg/ml of drug/gel at the backof the neck were at the same time injected with siHif1a_3 every otherday for 20 days beginning on day 0. As seen in FIG. 4E, ear hole closureinduced by 1,4-DPCA on day 14 and 20 was significantly inhibited bysiHif1a to a greater extent than even G₀ by itself, indicating the HIF1aspecificity of the regenerative effect on SW mice induced by 1,4-DPCAin-vivo, and a parallel to the HIF1a effect in spontaneouslyregenerating MRL mice.

Potential Mechanisms for HIF1a's Role in a Regenerative Response

The striking ability of amphibians to achieve regeneration is oftenattributed to the ability of cells to de-differentiate and become morestem-like before blastema growth. The effect of HIF1a stabilization wasexamined for appearance of stem cell markers in-vitro, as it waspreviously found that NANOG and SOX2 up-regulated inregeneration-competent adult MRL mice and that MRL ear-derivedfibroblasts in culture displayed multiple stem cell markers notexpressed in B6 cells. In FIG. 5A, MRL fibroblasts (5A, first row) arepositive for HIF1a and all immature markers tested; B6 fibroblasts (5A,second row) are negative, though rare positive staining was seen individing cells. However, B6 cells treated in-vitro with gel encapsulated1,4-DPCA for 24 hours (5A, third row) become positive displaying bothcytoplasmic and nuclear staining, indistinguishable from MRL cells withqPCR confirming these results. The B6 G₀ control was no different thanthe no-gel B6 cell control. QPCR results show highly significantdifferences between B6 fibroblasts treated with G₀ vs G_(d) for allmolecules tested (FIG. 5B). To address the specificity of HIF1a on thesemolecular markers in MRL, Hif1a siRNA-treatment (FIG. 5C) showed littleNANOG staining compared to control-treated MRL cells, demonstrating arequirement for Hif1a, at least for NANOG. SiHif1a was tested in B6cells after 1,4-DPCA treatment and also blocked multiple stem cellmarkers.

The role of HIF1a was explored in-vivo for multiple tissue phenotypesassociated with regenerating amphibian as well as MRL tissue (FIG. 6).Without limitation to any one theory or mode of operation,re-epithelialization, de-differentiation, remodeling, lowered collagencrosslinking, blastema growth and re-differentiation were among themechanisms for enhanced regeneration considered.

Very early and rapid re-epithelialization is a feature whichdistinguishes regeneration from wound repair. In the amphibian,re-epithelialization is complete within the first 12 hours and occursbetween 1-2 days in the MRL, but not until 5-10 days in the B6 and othermouse strains. Here, SW mice treated with drug/gel showedre-epithelialization by day 2 not seen in G₀-treated mice (FIG. 6Aa-b).Epidermal HIF1a expression is seen on day 1 and followed by WNT5aexpression on day 2 (FIG. 6. Ac-f), similar to drug-treated cellsin-vitro (FIG. 6Ag-i). This may contribute to rapid cell migration andwound coverage.

Paralleling the response in-vitro (FIG. 5A), de-differentiation is alsoseen in-vivo after a single injection of 1,4-DPCA drug/gel construct,with the expression of stem cell markers in the injured dermis (FIG.6Ba,b). NESTIN, OCT3/4, NF, and PAX7 expression by IHC (FIG. 6Bc-f) andqPCR (FIG. 6Bg) peaks at day 4-5 post-injury.

Tissue remodeling, including changes in extracellular matrix withbreakdown of basement membrane is necessary for axolotl limbregeneration. Laminin, a major component of the basement membrane, isreduced in SW ear tissue after 1,4-DPCA treatment (FIG. 6Ca-b), andabsent from the epithelial-mesenchymal border. MMP9, a major proteaseinvolved in laminin breakdown, increased after drug treatment (FIG. 6Cc)along with markers for inflammatory cells associated with tissueremodeling such as myeloperoxidase (MPO) and neutrophil-specific marker(Ly6G) (FIG. 6Cd-e) as well as mast cells.

Scarring, with increased collagen crosslinking, is associated with woundrepair and is reduced during amphibian regenerative responses. AfterG_(d) treatment, the picrosirius red (PSR) level, a marker of collagencross-linking complexity, is reduced, but reversed by siHif1a treatment(FIG. 6Da-d). Besides the expected reduced level of PHD function by1,4-DPCA, molecules such as loxl4 (lysyl oxidase-like 4) and Ctgf(connective tissue growth factor) are also reduced as shown by qPCR inSW ears after G_(d) treatment (FIG. 6De) similar to MRL.

Blastema growth and re-differentiation with chondrogenesis and hairfollicle growth, generally considered later events in the regenerativeprocess, are also affected by 1,4-DPCA. As seen previously in FIG. 4Band now in FIG. 6Ea-c, a major difference in ear hole size withaccompanying chondrogenesis and appearance of hair follicles (FIG.6Ee-h) is apparent by day 35 after G_(d) treatment of SW mice.Differences are seen histologically in tissue in the growth zone (FIG.6Ec) as well as the end of the cartilage at the site of the originalpunch margin (FIG. 6Ee-h) which show areas of condensation, formation ofa perichondrial region, and new chondrogenesis. Furthermore,chondrogenesis-associated molecules, Mgp, Itm2a, Matn3, Mia2, Col11,Prg4, Fmod, and Chad are upregulated on da21 in G_(d)-treated tissue(FIG. 6Ed). In addition, keratin (KRT)14-positive hair follicles arefound in G_(d) compared to G₀-induced tissue at a level seen in normalear tissue (FIG. 6Ej-k) with up-regulation of specific molecular markersincluding S100a4, Wif1, Dkk3, Dcn, and Tgfb2 (FIG. 6Ei).

A major function of HIF1 is the activation of angiogenic target genessuch as Vegf and an increase in angiogenesis. MRL cells andHIF1a-stabilized B6 cells, but not untreated B6 cells, were positive forvWF, an endothelial cell marker. It was also found that CD-31 positivecells and microvessels/capillaries were increased on day 7-post ear holeinjury in MRL ears compared to B6. The same was seen in SW-injured earsafter drug/gel treatment compared to control. RT-PCR demonstrates thatVegf and Hmox1 mRNA were up-regulated as well.

To test the requirement for angiogenesis in ear hole closure, endostatinwas administered to 1,4DPCA drug/gel-treated SW mice. Endostatin, likesiHif1a 3, showed almost complete blockage of ear hole closure in thesemice. However, siHif1a_3 and endostatin had differing effects on theexpression of molecular markers of stem cell state, angiogenic markers,tissue remodeling and inflammation, allowing a hierarchal ordering ofsome processes central to regeneration. On day 7 post-injury andtreatment, a near complete absence of HIF1a, CD31, and OCT ¾ stainingwas observed for both endostatin and siHif1a 3. A decrease in MMP-9,MPO, and a neutrophil specific-marker was found with siHif1a_3 but to alesser degree with endostatin. On the other hand, a greater degree ofinhibition of laminin staining was found with endostatin than withsiHif1a_3.

Following its discovery by Semenza, there has been a growing recognitionthat HIF1a is a master regulator of cell functions from regulating O₂levels to aerobic glycolysis, cell migration and inflammation. In thisreport, we propose another role for HIF1a, i.e. as a central actor inmammalian regeneration. Given HIF1a's many known functions in cellularprocesses that distinguish tissue regeneration from a scarring (tissuerepair) response, it was natural to explore HIF1a's role in regenerativewound healing in the MRL mouse, a strain which uses aerobic glycolysisas its basal metabolic state and is a spontaneous regenerator ofmultiple tissue types. Furthermore, a recent genetic fine mapping studyshowed that RNF7, an E3 ligase necessary for HIF1a ubiquination, is astrong candidate gene for LG/J ear hole regeneration, is down-regulatedin both LG/J and MRL mice, and should predictably lead to high HIF1alevels. As shown in the results, HIF1a is upregulated in unwounded MRLversus B6 and SW mice, is further increased post-wounding, and siHif1ablocks MRL ear hole closure. To determine the effect of high levels ofHIF1a in non-regenerative Swiss Webster mice, the HIF1a-stabilizing drug1,4-DPCA was delivered subcutaneously via a hydrogel construct, inducingear hole closure when given both proximally and distally. As in MRL,in-vivo siRNA against Hif1a blocks this drug-induced regenerativeresponse supporting the conclusion that, at least in mice, up-regulationof HIF1a levels is sufficient to achieve appendage regeneration.

Creating an effective delivery system for 1,4-DPCA presented a majorchemical challenge. A problem is the low solubility of this moleculeand, hence, the inability to deliver a biologically effective dosing.This was overcome by the use of block copolymer-stabilized 1,4-DPCAmicrocrystals embedded in a hydrogel. In-vitro results usingdifferentiation and other markers led to the belief that this was apromising in-vivo approach. A regenerative response identical to thatobserved in MRL mice was achieved using this construct.

Recent data has shown that two inhibitors of PHDs which block HIFdegradation, DMOG and DFX, enhance diabetic wound healing when applieddirectly to the wound site. However, the induction of a regenerativeresponse requires far higher levels of HIF1a and has not been reported.Results reported herein describe a novel structure and method of drugdelivery using a drug crystal and hydrogel that may be effective totreat injury through a systemic route. The idea of inducing tissueregeneration via a simple, minimally invasive subcutaneousadministration of drug carrier at a peripheral site is both attractiveand a significant departure from previous tissue regeneration paradigms.

The use of 1,4-DPCA-hydrogels has several advantages. They slowlydeliver large amounts of 1,4-DPCA from the hydrogel over 4-10 daysdepending on the gel formulation and drug dose when tested in-vitro, andcause increased HIF1a protein levels in-vivo for up to 5 days. Also,entrapping the drug microcrystals within the hydrogels avoids potentialcytotoxicity associated with direct uptake of drug crystals by cells. Interms of specificity, 1,4-DPCA interacts with and blocks PHD functionand could affect not only HIF1a, but HIF2a as well as other targetmolecules. However, HIF2a is not affected by the drug gel constructin-vitro, in either fibroblasts or endothelial cells, nor is itincreased in ear tissue after G_(d) treatment. Furthermore, siHif1ablocked all regenerative phenotypes examined.

Limb regeneration in amphibians generally centers on the formation andgrowth of the blastema, a tissue structure seen in the embryo andregenerating tissue and made up of a mass of undifferentiatedpluripotent cells which can proliferate and then produce a copy of thelost structure. This begins with rapid coverage of the wound byepithelial cells, reforming in the absence of a basement membrane.Undividing mesenchymal cells form under the new epidermis as theaccumulation blastema and then divide, produce tissue elongation, andfinally re-differentiate into lost parts.

HIF1a-regulated gross regenerative effects in MRL and drug-treated SWmice emulates molecular and cellular correlates of the amphibianblastema and de-differentiation followed by tissue remodeling andproliferation and later followed by re-differentiation components of theclassical regeneration process. HIF1a is expressed at its highest levelsin the early phase of the regenerative response. This is associated withthe accumulation blastema period forming through cell migration andde-differentiation in regenerating tissue and is consistent withup-regulation of molecules such as WNT5a involved in cell migration, andNANOG and OCT3/4 as de-differentiation markers peaking at approximatelyday 7 post-injury, after which the levels of these molecules fall andproliferation proceeds. The developmental state occurs in low levels ofoxygen. This hypoxic state results in increased HIF1a, increasedmorphogenesis, and increased presence of stem cells with the inductionof multiple ESC-associated genes and differentiation markers. MRL eartissue showed unusual expression of a range of diverse stem cell markersboth in-vitro and in-vivo including NANOG, SOX2, OCT3/4, CD34, andCD133, all pluripotency markers; NESTIN, a neuronal stem and progenitorcell marker; PAX7, a satellite muscle-associated stem cell marker;WNT5a, an early marker involved with migration, and PREF1 or DLK1, apre-adipocyte and hepatocyte stem cell marker. This was not found innon-regenerator B6 or SW tissue. HIF1a stabilization by 1,4-DPCA led toincreased levels of all of these differentiation markers, though onlytransiently, reducing long-term concerns associated with potentialtreatment. SiHif1a blocked NANOG expression in MRL cells suggesting thatall of these markers are due to increased HIF1a levels in this mouse;siHif1a blocked many of these markers in 1,4-DPCA+siHif1a-treatednon-regenerative cells and tissues. Besides a hypoxic environment andelevated HIF1a, stem cells require a glycolytic metabolism seen in MRLmice and other regenerating models. Surprisingly, HIF2a, reported tocontrol expression of Nanog and Oct3/4, is not elevated, though a recentstudy shows other controlling factors such as miR-302.

To further confirm that this drug-induced regenerative responsefaithfully emulates the phenomena observed in the MRL mouse, and keyprocesses observed for many years in classical regenerators such asnewts and axolotls, other known HIF1a functions were examined. Theseinclude an enhanced tissue remodeling response, increased MMP levels, ade-differentiated cellular signature, increased glycolytic enzymes,increased components of the inflammatory response and increasedangiogenesis leading to ear hole closure.

Next, tissue remodeling necessary for ECM changes in regeneratingamphibian limb blastemas shows increased MMP levels and no basementmembrane which if restored using retinoic acid treatment produces scarwith no regeneration. HIF1a regulates MMPs which regulate extracellularmatrix levels including laminin and basement membrane-remodelingproteins. Like MRL, 1,4-DPCA-treated SW mice show increased MMP9 levelsand a vanishing basement membrane (FIG. 6Ca,b). A secondregeneration-promoting effect of 1,4-DPCA on PHDs is inhibition ofcollagen hydroxylation leading to reduced scarring and increaseddegradation. Other HIF1a-regulated remodeling molecules include lysyloxidase and collagen prolyl (P4HA1,2) and lysyl (PLOD2) hydroxylaseswhich increase ECM stiffness and alignment. MRL and 1,4-DPCA-treated SWmice express reduced levels of loxl4 and ctgf.

The developmental state occurs in low levels of oxygen and this hypoxicstate results in increased HIF1a, increased morphogenesis, and increasedstem cells with the induction of multiple ESC-associated genes anddifferentiation markers. MRL ear tissue showed unusual expression of arange of diverse stem cell markers both in-vitro and in-vivo includingNANOG, SOX2, OCT3/4, CD34, and CD 133, all pluripotency markers; NESTIN,a neuronal stem and progenitor cell marker; PAX7, a satellite muscleassociated stem cell marker; WNT5a, an early cell marker; and PREF1 orDLK1, a pre-adipocyte and hepatocyte stem cell marker. This was notfound in non-regenerator mouse tissue from either B6 or SW.

However, HIF stabilization by 1,4-DPCA led to increased levels of all ofthese differentiation markers, though only transiently, making this lessof a concern with treatment. SiHif blocked NANOG expression in MRL cellssuggesting that perhaps all of these markers are due to increased HIF1alevels in this mouse and siHif blocked OCT3/4 post 1,4-DPCA treatment.Besides an hypoxic environment and elevated HIF1a, stem cells require aglycolytic metabolism, seen in the MRL mouse and other regeneratingmodels. Though it is not clear why this metabolic state is necessary,Cripto/GRP78 may play a role. The virtual identity of all of the abovemarkers in MRL ear cells and the 1,4-DPCA-treated SW cells dramaticallyconfirms the unity of a regeneration-type response in these models.

Re-differentiation of mesenchymal tissue with formation of new cartilageand hair follicles is seen in regenerating tissue. In MRL, elastic andarticular cartilage begins at about 1 month and can fully regeneratewithin 3-4 months. With 1,4-DPCA treatment, the new growth area showschondrogenesis by day 35, with upregulation of multiple chondrogenesismarkers including those in chondrogenic precursor cells and moleculesfound in cartilage extracellular matrix. Hair follicles are also foundin the new growth area at a level seen in normal tissue and multiplemarkers of bulge-derived keratinocyte stem cells and cells in theepithelial sac involved in regeneration are expressed.

Down-regulation of HIF1a inhibits inflammation. It has been shown usinga Hif1a conditional knockout mouse that HIF1a is required and controlsthe inflammatory response through regulation of glycolysis, a statenecessary for myeloid (including neutrophils and macrophages) survivaland function with effects specifically on aggregation, invasion,motility, and cutaneous inflammation. NSAIDs such as the COX2 inhibitorsindomethacin, meloxicam and ibuprofen, which negatively regulateinflammation, also inhibit HIF1a through the up-regulation of pVHLexpression. Down-regulation of HIF1a in a Hif KO mouse has been shown toheal burn wounds poorly with a concomitant reduction in angiogenesis andSDF1. As shown here, MPO, a marker of inflammation, and a neutrophilspecific marker are up-regulated in the non-regenerative SW given1,4-DPCA similar to the MRL mouse.

Previous studies showed the role of angiogenesis in the regenerativeresponse such as an AGF (angiopoietin-related growth factor) tg mousewith increased vascular and epithelial proliferation and positive earhole closure and an angpt1 (angiopoetin1) ko mouse (angpt1 negativelyregulates angiogenesis) with positive ear hole closure, and the knowneffects of HIF1a on angiogenesis. Comparing endostatin, a broad spectrumangiogenesis inhibitor, to siHif for their ability to block 1,4DPCA-induced hole closure, it was found that all of the phenotypesdescribed above were affected by siHIF and endostatin, some similarlyand some oppositely allowing us to generate a preliminary map ofeffector function.

Three molecules, HIF1a, CD31, and OCT3/4, were inhibited equally bysiHif and endostatin. This is to be expected for CD31 which reacts withendothelial cells and vessels as well as for HIF1a which has beenpreviously shown to be down-regulated by endostatin. However, the equalblocking of OCT3/4 expression with endostatin treatment suggests thatOCT3/4 is downstream of endostatin and angiogenesis. In contrast, unlikesiHIF, endostatin did not inhibit MPO and anti-neutrophil marker(markers of inflammation) or MMP9 (remodeling) suggesting that theseeffector functions are upstream of angiogenesis and stemcells/de-differentiation.

Finally, laminin expression was most affected by endostatin which wassurprising as it is considered to be involved with remodeling. However,it is consistent with laminin expression in blood vessels which whenvessel formation is blocked should reduce laminin levels.

These data, taken together, as highlighted by the virtual identity ofall of the differentiation markers in both MRL ears and 1,4-DPCA-treatedB6 cells as well as drug-treated SW ears strongly supports the unity ofthe spontaneously regenerating MRL mouse model, the 1,4-DPCA-inducedregeneration in SW mice and classical amphibian regenerators observed innature.

EXAMPLES OF THE INVENTION

The following non-limiting examples and data illustrate various aspectsand features relating to the methods and/or systems of the presentinvention, including the preparation of various hydrogels as areavailable through the synthetic methodologies described or referencedherein. In comparison with the prior art, the present methods andsystems provide results and data which are surprising, unexpected andcontrary thereto. While the utility of this invention is illustratedthrough the use of several hydrogel systems and precursor components,and PHDs which can be used therewith, it will be understood by thoseskilled in the art that comparable results are obtainable with variousother hydrogel systems, precursor components and therapeutic agents, asare commensurate with the scope of this invention.

Inbred mouse strains were used to study the effect of a small moleculeinhibitor of PHD on in-vitro and in-vivo levels of HIF1a, and the impactof this on quantitative regenerative ear hole closure phenotypes. Inanimal studies, 2.1 mm ear hole punch wounds were created and a1,4-DPCA-containing hydrogel was subcutaneously implanted in the back ofthe neck of mice at multiple time-points. Healing was monitored bymeasuring hole diameters. Endpoints of the study were previouslydetermined to be 30+ days post-injury and included key indices of tissueregeneration such as blastema formation, epithelial, dermal, andcartilaginous wound closure with hair follicle replacement plus multiplemolecular markers of cellular de-differentiation, re-differentiation,and stem cell state. These parameters were determined by physicalmeasurements of wound closure, standard tissue histology andhistomorphometry, and gene expression using quantitativeimmunohistochemistry, western analysis, and qPCR. The experimentalgroups were coded and different laboratory personnel were involved ininjuries, injections, phenotyping and data analysis.

Example 1 Animals and In-Vivo Procedures

MRL/MpJ and Hif1a ODD-luciferase reporter(FVB.12956-Gt(ROSA)26Sortm2(HIF1A/luc)Kael/J) mice were obtained fromJackson Laboratories (Bar Harbor, Me.); C57BL/6 (B6) mice were fromTaconic Laboratories (Germantown, N.Y.); Swiss Webster (SW) mice werefrom Charles River (New York, N.Y.). Mice were used at approximately8-10 weeks in all experiments under standard conditions at the WistarInstitute Animal Facility (Philadelphia, Pa.) and the protocols were inaccordance with NIH Guide for the Care and Use of Laboratory Animals.Through-and-through ear hole punches were carried out as previouslydescribed.

Example 2 IVIS Luciferase Scanning

To detect luciferase expression in-vivo, mice were given a single i.p.injection of D-luciferin (37.5 mg/kg, Gold Biotechnology Inc) in sterilewater. Fifteen minutes later, mice were anesthetized using isofluraneand placed in a light-tight chamber equipped with a charge-coupleddevice IVIS imaging camera (Xenogen, Alameda, Calif.). Photons werecollected for a period of 1-5 min, and images were obtained by usingLIVING IMAGE software (Xenogen) and IGOR image analysis software(WaveMatrics, Lake Oswego, Oreg.). HIF1a ODD luc expression after earpunching was determined in MRL and B6 mice backcrossed to the transgenicHIF1a-peptide-luciferase reporter mouse FVB.129S6-Gt(ROSA)26S, made byfusing luciferase to the domain of HIF1a that binds to pVHL in aoxygen-dependent way (ODD peptide) mice and selected for luciferasepositivity.

Example 3 Tissue Culture

Primary ear dermal fibroblast-like cells were established from MRL andB6 mice and grown in DMEM-10% FBS supplemented with 2 mM L-glutamine,100 IU/mL penicillin streptomycin and maintained at 37° C., 5% CO₂, and21% O₂. Cells were split 1:5 as needed to maintain exponential growthand avoid contact inhibition. Passage numbers were documented and cellsfrom early passages (<P20) frozen in liquid nitrogen and used in thedescribed experiments.

Example 4 Western Analysis

Ear tissue samples (3 ear hole donuts/ear from 3 separate mice) werehomogenized in radio-immunoprecipitation assay buffer (50 mM Tris-HCl pH7.6, containing 150 mM NaCl,1% Triton X-100, 1% sodium deoxycholate, 1mM EDTA and 0.1% SDS) with 1 mM PMSF and a protease inhibitor cocktail(Sigma). Samples with equal amounts of protein (about 40 μg) were loadedinto a NuPAGE 4-12% Bis-Tris gradient gel or 8% Bis-Tris gel (LifeTechnologies, Grand Island, N.Y.), electrophoresed and thenelectro-transferred onto a PVDF-FL membrane (Immobilon, Billerica,Mass.). The membrane was subsequently blocked with Odyssey blockingbuffer (LI-COR, Lincoln, Nebr.), probed with primary antibodies (HIF1a(10006421, Cayman Chemical, Ann Arbor, Minn.), HIF2a (NB100-132B, Novus,Littleton, Colo.), Wnt5a (BAF645, R&D System) or a-Tubulin (Sigma)overnight at 4° C., then further incubated with Alexa Fluor-labeledsecondary antibodies (IRDye 800CW goat-anti rat or IRDye 800CW goat-antirabbit (LI-COR, Lincoln, Nebr.) for 1 hr and scanned using the Odysseysystem (LI-COR, Lincoln, Nebr.).

Example 5 Hif-1a siRNA Transfection In Vitro and In Vivo

B6, SW, and MRL ear fibroblast-like cells at 70% of confluence weretransfected with 100 nM of 4 different HIF-1a siRNAs (SI00193025,SI00193032, 5100193011, SI00193018) purchased from Qiagen and scramblesiRNA (sc-37007, Santa Cruz Biotechnologies), using Lipofectamine 2000according to the manufacturer's protocol. Transfected cells wereexamined for the knockdown efficiency after 48 h of transfection. siRNAMm_Hif1a_3 (SI00193025) was selected for the in vitro experiments due toits high efficiency. In vivo, siRNA Mm_Hif1a_3 was used for HIF-1ainhibition. SiHif at 75 mg/kg body weight was mixed with Jetpei(Polyplus, Genycell) following manufacturer's instructions and was theninjected into animals subcutaneously every 48 h.

Example 6 RNA Isolation and RT-PCR

Total RNA from ear fibroblast-like cells or ear hole donuts was preparedwith Qiagen RNeasy kit (Qiagen) according to the manufacturer'sguidelines. First strand cDNA was synthesized from 1 μg of RNA using theSuperscript First-Strand Synthesis System (Invitrogen, Carlsbad, Calif.)according to the manufacturer's instructions. qPCR was performed withSYBR green PCR Master Mix (Applied Biosystems, Life Technologies). Inbrief, a 20 μl mixture was used containing 10 μl SYBR Green PCR mastermix, 1 μl forward and reverse primer, 6 μl sterile water, and 2 μl ofcomplementary DNA template. A negative control (non-template control)was performed in each run. The real-time PCR was performed using a QuantStudio 6 Flex (Applied Biosystems) according to the manufacturer'sinstructions. All data were normalized to 18S rRNA and quantitativemeasurements were obtained using the ΔΔC_(T) method. (The primers usedare listed with the kit, but not shown, here.

Example 7 Immunohistochemistry

The methods used were performed as previously described. Tissue fromnormal ears were fixed with Prefer fixative (the active ingredient isglyoxal) (Anatech) overnight and then washed in H₂O. Tissue was embeddedin paraffin and 5-μm thick sections cut. Before staining, slides weredewaxed in xylene and rehydrated. Antigen retrieval was performed byautoclaving for 20 min in 10 mM Sodium Citrate, pH 6.0. Tissue sectionswere then treated with 3% H₂O₂ and nonspecific binding was blocked with4% BSA (A7906; Sigma) for 1 h. The primary antibodies and matchedsecondary antibodies used for IHC were shown in Table 2. Forimmunocytochemistry staining, primary ear skin fibroblasts were grown oncoverslips in DMEM with 10% FBS at 37° C. in a humidified 5% CO₂incubator. The coverslips were rinsed with 1×PBS, the cells were fixedin cold methanol (−20° C.) for 10 min, rinsed with 1×PBS, treated with0.1% Triton-X100, and then incubated with the appropriate primary andsecondary antibodies (Table 1). Photomicrographs were produced using thefluorescent microscope (Olympus AX70) and a Spot camera with bundledsoftware.

For histological stains, tissue sections were treated the same as aboveand then stained with Hematoxylin (Leica Microsystems, #3801562) andEosin (Leica Microsystems, #3801602), Picro-Sirius Red (Poly Scientific,cat. # s2365), Alcian blue (1% in 3% acetic acid (Polyscience, BayShore, N.Y., cat # S111A), or toluidine blue O (Allied Chemical,Morristown, N.J., cat. # NA0652), counterstained with Kernechtrot(Polyscience, Bay Shore, N.Y., cat # S248). The slides were washed,rehydrated, cleared with Xylene and coverslipped with Permount mountingmedia (Fisher, SP15-500). Staining was visualized using an Olympus(AX70) microscope in bright field for H&E and under polarized light forPicro-Sirius Red.

For quantitation of IHC signal, the method used was previouslydescribed. Briefly, we used ImagePro v4.0 for image analysis byselecting positive staining from multiple areas in the sections. Thenumber of “positive staining” pixels was determined. The area wasexpressed in square microns and the final data were expressed as IHCstaining signal per square micron. The mean of 2-6 samples were plottedand standard errors calculated.

TABLE 1 Antibodies used for Immunostaining 1st antibody 2nd antibody AllFrom Molecular Company Cat. no. Dilution Probe Company Cat. no. DilutionHIF1a Abcam ab2185  1:1000 Alexa Fluor 488 Molecular Probe A11008 1:200goat anti-rabbit IgG Nanog Calbiochem SC1000 1:150 Alexa Fluor 568Molecular Probe A11036 1:400 goat anti-rabbit IgG Oct-3/4 Santa Cruzsc-5279 1:150 Alexa Fluor 568 Molecular Probe A11061 1:400 rabbit anti-mouse IgG CD133 Chemicon MAB4310 1:100 Alexa Fluor 594 Molecular ProbeA11007 1:200 goat anti-rat IgG CD34  Bioss bs-0646R 1:200 Alexa Fluor568 Molecular Probe A11036 1:300 goat anti-rabbit IgG Wnt5a R&D BAF6451:150 Alexa Fluor 568 Molecular Probe A11057 1:200 Systems donkeyanti-goat IgG PAX7 R&D MAB1675 1:50  Alexa Fluor 568 Molecular ProbeA11061 1:400 Systems rabbit anti- mouse IgG Pref-1 MBL D187-3 1:10 Alexa Fluor 594 Molecular Probe A11007 1:200 International goat anti-ratIgG Nestin DHSB 1:50  Alexa Fluor 594 Molecular Probe A11005 1:200 goatanti-mouse IgG vWF Dako A0082 1:100 Alexa Fluor 488 Molecular ProbeA11008 1:200 goat anti-rabbit IgG Neth Sigma N0142 1:200 Alexa Fluor 568Molecular Probe A11061 1:400 rabbit anti- mouse IgG Lamc2 Sigma L-93931:50  Alexa Fluor 568 Molecular Probe A11036  1:1000 goat anti-rabbitIgG MPO NeoMarkers RB- 1:70  Alexa Fluor 488 Molecular Probe A110081:200 373A1 goat anti-rabbit IgG Anti- Cedarlane CL8993F 1:40  FITCmouse Jackson 212096082 1:100 Neutrophil anti-rat IgG ImmunoResearch mAblab MMP9 Sigma M9555 1:200 Alexa Fluor 568 Molecular Probe A11036 1:200goat anti-rabbit IgG

Example 8 Data Analysis

All experiments were repeated multiple times (N) and the data representpooled samples for western analysis and qPCR, and individual samples inhealing studies and tissue analysis (n) as indicated in figure legends.All experiments employed inbred mouse strains reducingindividual-to-individual variation. Student's t-test was carried out tocompare differences of means from independent samples between twogroups. The ANOVA test was performed to determine if there weresignificant differences among the means of more than two groups. If thep-value from ANOVA analysis was significant, then the post-hoc Tukeytest was applied to compare the mean between each group. P-values lessthan or equal to 0.05 were considered as significant and equal or lessthan 0.01 considered highly significant. All error bars shown on graphsrepresent standard errors (SE) except in FIG. 2 where the standarddeviation (SD) is used. The software used for ANOVA analysis and thepost-hoc Tukey test is R, version 2.14.1. All other statistical analyseswere done using Microsoft Excel 2010.

Example 9

With reference to FIG. 7, the compound 1,4-DPCA was prepared as follows:

Example 9a Synthesis ofDiethyl[(quinolin-8-ylamino)methylidene]propanedioate (1)

1 was prepared using a modified protocol described previously in theliterature. 8-aminoquinoline (3.31 g, 22.9 mmol) and diethylethoxymethylenemalonate (4.63 mL, 22.9 mmol) were heated to 100° C. for1 hour and then cooled to 80° C. and 20 mL methanol was added. Thecrystallized product was washed twice with 20 mL MeOH and dried on highvacuum to afford 1 (5.68 g, 18.1 mmol, 79%) as green-brown needles. ¹HNMR (500 MHz, Chloroform-d), δ, ppm (J, Hz): 12.37 (1H, d,³J_(NH,−CH)=14.3, NH); 8.97 (1H, dd, ³J_(2,3)=4.3, ⁴J_(2,4)=1.7, H-2);8.80 (1H, d, ³J=_(CH,NH)=14.3, ═CH); 8.18 (1H, dd, ³J_(4,3)=8.3,⁴J_(4,2)=1.7, H-4); 7.55 (3H, m, H-5,6,7); 7.49 (1H, dd, ³J_(3,4)=8.3,³J_(3,2)=4.2, H-3); 4.42 (2H, q, ³J_(OCH2,cH3)=7.1, (Z)-ester OCH₂);4.30 (2H, q, ³J_(OCH2,CH3)=7.1, (E)-ester OCH₂); 1.44 (3H, t,³J_(CH3,OCH2)=7.1, (Z)-ester CH₃); 1.37 (3H, t, ³J_(CH3,OCH2)=7.1,(E)-ester CH₃).

Example 9b Synthesis of Ethyl4-oxo-1,4-dihydro-1,10-phenanthroline-3-carboxylate (2)

2 was prepared using a modified protocol described previously. 1 (5.50g, 17.5 mmol) was added to diphenylether (55 mL) and refluxed (250° C.)for 1 hour, then cooled to room temperature and collected throughfiltration. The precipitate was triturated twice with 25 mL petroleumether (b.p. 80-110° C.) followed by washing with 10 mL of cold Et₂O. Theprecipitate was dried on high vacuum overnight to afford 2 (2.05 g, 7.65mmol, 44%) as a beige powder. ¹H NMR spectrum (500 MHz, DMSO-d₆), δ ppm(J, Hz): 12.88 (1H, s, NH); 9.10 (1H, dd, ³J_(2,3)=4.3, ⁴J_(2,4)=1.6,H-2); 8.57 (1H, dd, ³J_(4,3)=8.3, ⁴J_(4,2)=1.6, H-4); 8.54 (1H, s, H-8);8.22 (1H, d, ³J_(6,5)=8.8, H-6); 7.90 (1H, d, ³J_(5,6)=8.8, CH, i); 7.84(1H, dd, ³J_(3,4)=8.3, ³J_(3.2)=4.3, H-3); 4.25 (2H, q,³J_(OCH2,CH3)=7.1, OCH₂); 1.30 (3H, t, ³J_(CH3,OCH2)=7.1, CH₃).

Example 9c Synthesis of 1,4-dihydrophenonthroline-4-one-3-carboxylicacid (1,4-DPCA) (3)

3 was prepared using a modified protocol described previously. 2 (2.00g, 7.46 mmol) was combined with 40 mL 10% (w/v) KOH and refluxed (110°C.) for 1 hour, allowed to cool to room temperature, and residualdiphenyl ether extracted using 28 mL petroleum ether (b.p. 80-110° C.).The product was precipitated with 40 mL 10% (w/v) HCl, filtered, washedwith dH₂O and dried under high vacuum overnight to afford 3 (1.65 g,6.87 mmol, 92%) as a beige powder. ¹H NMR spectrum (500 MHz, DMSO-d₆),δ, ppm (J, Hz): 15.44 (1H, s, OH); 13.85 (1H, s, NH); 9.16 (1H, dd,³J_(2,3)=4.3, ³J_(2,4)=1.6, H-2); 8.73 (1H, s, H-8); 8.64 (1H, dd,³J_(4,3)=8.3, ⁴J_(4,2)=1.6, H4); 8.26 (1H, d, ³J_(6,5)=8.8, H-6); 8.04(1H, d, ³J_(5,6)=8.8, H-5); 7.92 (1H, dd, ³J_(3,4)=8.3, ³J_(3,2)=4.3,CH, k). Mass spectrum, m/z (I_(rel), %): 241.1 [MH]+(18), 263.0 (100),279.0 (30), 503.1 (41). Purity was estimated as 99.8% by HPLC (C₁₈, 10μm, 4.6×250 mm, 300 Å pores, silica; 2-100%, 30 min, acetonitrilegradient, 0.1% TFA; elution time=18.5 minutes; UV-vis, λ_(max), nm: 261,316, 331, 346).

Example 10

With reference to FIG. 2, a hydrogel carrier component and precursorsthereto are described in Examples 10a-c. Various other hydrogel carriersand precursors, in accordance with this invention are described inco-pending application Ser. No. 13/798,744 filed Mar. 13, 2013, or aswould otherwise be understood by those skilled in the art made aware ofthis invention through straight forward modifications of the synthetictechniques described therein—such application incorporated herein byreference in its entirety.

Example 10a Synthesis of Glutaric Acid Terminated 8 Arm PEG (P8G)

Glutaric acid terminated PEG was synthesized as described previously.Briefly, 8-arm PEG-OH (19.4 g, 7.74 mmol OH) and glutaric anhydride(4.49 g, 38.7 mmol) were dissolved in chloroform (20 mL). Pyridine (3.12mL, 38.7 mmol) was added dropwise, and the reaction mixture was refluxedat 82° C. for 24 hours under inert air. The product was precipitatedwith cold diethyl ether (200 mL) and spun down. The supernatant wasdecanted and the product re-dissolved in MeOH (200 mL). After incubationat −20° C. for 1 hour, the precipitate was centrifuged at −5° C. Thesupernatant was discarded, and the MeOH wash procedure was repeatedtwice more. Following cold diethyl ether precipitations (400 mL), theproduct was collected and dried under high vacuum overnight to afford awhite powder (92% yield, 100% conversion). 1H NMR (500 MHz,Chloroform-d), δ, ppm: 4.24 (16H, t, terminal PEG CH2), 3.64 (1823H, m,backbone PEG CH2), 2.43 (16H, t, H-2 of glutaric acid), 2.39 (16H, t,H-4 of glutaric acid), 1.96 (16H, p, H-3 of glutaric acid).

Example 10b Synthesis of N-Hydroxysuccinimide Terminated 8 Arm PEG(P8NHS)

NHS terminated 8 arm PEG was synthesized as described previously. P8G(18.6 g, 7.10 mmol COOH), NHS (8.18 g, 71.0 mmol) and EDC (13.6 g, 71.0mmol) were dissolved in DMSO (47 mL). The solution was agitated for 30minutes at room temperature, then it was diluted with MeOH (200 mL),precipitated at −20° C. for 1 hour, and spun down at −5° C. Thesupernatant was decanted, and the MeOH wash procedure was repeated twicemore with 400 mL MeOH per wash. Following cold diethyl etherprecipitations (400 mL), the product was dried under high vacuum toafford a white powder (95% yield, 96% conversion). 1H NMR (500 MHz,Chloroform-d), δ, ppm: 4.24 (16H, t, terminal PEG CH₂), 3.63 (1823H, m,backbone PEG CH₂), 2.84 (32H, m, NHS protons), 2.71 (16H, t, H-4 ofglutaric acid), 2.49 (16H, t, H-2 of glutaric acid), 2.06 (16H, p, H-3of glutaric acid).

Example 10c Synthesis of Cysteine Terminated 8 Arm PEG (P8Cys)

Cysteine terminated 8 arm PEG was synthesized as described previously.PEG-NH₂ (20 g, 8.12 mmol NH₂) was dissolved in DMF (40 mL) after whichDIEA was added dropwise (1.41 mL, 8.12 mmol). In a separate reactionvessel, Boc-Cys(Trt)-OH (15.0 g, 32.5 mmol) and BOP (14.4 g, 32.5 mmol)were dissolved in DMF (40 mL) and DIEA (5.65 mL, 32.5 mmol) was addeddropwise. Both solutions were combined, and the coupling reaction wasallowed to proceed at room temperature for 18 hours. Followingprecipitation in cold diethyl ether (400 mL), the product wasre-dissolved in MeOH (40 mL) and precipitated in cold diethyl ether oncemore (400 mL). The cysteine was deprotected with TFA:TIS:EDT (300 mL,95:2.5:2.5) cleavage solution at room temperature for 4 hours. TFA wasevaporated under low pressure, and the product was precipitated in colddiethyl ether (400 mL). P8Cys was dissolved in MeOH (200 mL),precipitated at −20° C. overnight, and centrifuged at −5° C. Thesupernatant was decanted and the MeOH precipitation was repeated twicemore using 100 mL MeOH per wash. Following diethyl ether precipitations(200 mL), the product was dried under high vacuum overnight to afford awhite powder (73% yield, 84% endgroup conversion). 1H NMR (500 MHz,Acetic Acid-d4), δ, ppm: 4.41 (8H, t, α-C cysteine), 3.68 (1790H, m,backbone PEG CH2), 3.13 (16H, d, CH2 cysteine).

Example 11 Preparation of 1,4-DPCA/F127NF Crystals

1.35 g Pluronic F127NF and 100 mg 1,4-DPCA were dissolved in 10 mL DMF.With stirring, the F127NF/1,4-DPCA solution was added dropwise to 500 mLddH₂O at 60° C. The resulting crystals were collected by filtration andwashed twice with 200 mL 0.27% (w/v) F127NF in ddH₂O. The crystals werere-suspended in 50 mL 0.27% (w/v) F127NF in ddH₂O and lyophilized toafford a white powder of F127NF/1,4-DPCA drug crystals (DCs). HPLC (C₁₈,10 μm, 4.6×250 mm, 300 Å pores, silica; 2-100%, 30 min, acetonitrilegradient, 0.1% TFA; elution time=18.5 minutes) was used to quantify thedrug content. The amount of 1,4-DPCA in the drug microcrystals showedbatch to batch variation within the range of 35-53%.

Example 12 Filter Sterilization of PEG Polymers

A 10% (w/v) solution of each PEG polymer in MeOH was filtered through a0.2 μm filter into a sterile receptacle. The product was lyophilized toyield filter sterilized P8NHS or P8Cys.

Example 13 Formation of Drug-Loaded Hydrogels

Separately, 10% (w/v) solutions of P8NHS and P8Cys were prepared inphosphate buffered saline (PBS) suspension of DCs at the desired drugconcentration. The two polymer solutions were then mixed in a 1:1 v/vratio and left undisturbed for 20 minutes to yield 70 μL cylindricalhydrogels (n=3).

Example 14 In-Vitro Drug Release from Hydrogels

Each hydrogel cylinder prepared as described above was immersed in 5 mLPBS and at specified time points transferred into 5 mL of fresh PBS.UV/vis spectrophotometry was used to quantify drug release over time.The standard curve was prepared from stock solutions of knownconcentration of drug in DMSO. 10 μL of each stock solution was added to990 μL PBS to yield a standard curve with 100-3000 ng/mL of drug. APowerWave XS2 microplate spectrophotometer (Biotek Instruments, Inc.,Winooski, Vt., USA) was used to quantify absorbance at 261 nm. Hydrogelswithout drug were used as negative controls.

Example 15 In-Vivo Hydrogel Injection

Mice were injected subcutaneously at the base of the neck with 100 μL of10% (w/v) 1:1 (w/w) ratio of P8Cys (with or without 1,4-DPCA drugcrystal) to P8NHS hydrogel prepared in PBS. Each component was kept coldand mixed just prior to injection. At different time points, mice wereeuthanized and tissues were removed for protein and RNA analysis.

Example 16 Gelation Kinetics

Gelation time was quantified using a previously described protocol.Briefly, the drug microcrystals were suspended in PBS and used toprepare 10% (w/v) P8NHS and 10% (w/v) P8Cys. The two polymer solutionswere then mixed in a 1:1 (v/v) ratio and pipetted up and down using astandard 0.1-10 μL pipette tip. The time at which the material blockedthe pipette tip was designated as the gelation time. Temperature wascontrolled at 37° C. through the use of a water bath.

Example 17 Cell Viability

Viability of 3T3 fibroblasts exposed to drug microcrystals wasquantified using ISO 10993. Briefly, different dilutions of drugmicrocrystals in cell culture medium were added to a subconfluentmonolayer of 3T3 fibroblasts (n=3). The cells were cultured for 24 hoursat 37° C., 5% CO₂, and >90% RH and then washed with PBS. Neutral redsolution (0.4%) in DMEM was added, and the cells were stained for 3hours. Following removal of the staining solution and washing the cellswith PBS, the cells were destained using 50% ethanol, 49% ddH₂O and 1%glacial acetic acid. Following 10 minutes of agitation, absorbance wasmeasured at 540 nm and used to quantify cell viability. SDS was used asa positive control as specified by ISO 10993, and the IC₅₀ was found tobe in the acceptable range of values hence confirming the validity ofthe assay. Culture medium was used as a negative control. Cell culturemedium consisted of high glucose DMEM substituted with L-glutamine,penicillin/streptomycin, 10% v/v newborn calf serum and 20 mM HEPES.

Example 18

As an alternative to the drug conjugate of Example 11, 1,4-DPCA wascoupled to 8 arm PEG to provide another route to or system for delivery(see FIG. 8A and Examples 18a-b). The drug was released via esterhydrolysis with approximately 35% hydrolysis over a 20 day period (seeFIG. 8B and Example 18c).

Example 18a Synthesis of DPCA-I

1,4-DPCA (1 g, 4.163 mmol) was mixed with 22.2 mL DMF and stirred for afew minutes at room temperature. 1,1′-carbonyldiimidazole (1.69 g,10.408 mmol) was added to the mixture with heating at 100° C. for 2.5hours. The rxn vessel was removed from heat and allowed to reach roomtemperature. The precipitate was collected by filtration and washedtwice with 10 mL chloroform. The product was dried under high vacuum toyield 1.169 g DPCA-I (96.7%) with approximately 96.2% activation/purity.¹H NMR (500 MHz, DMSO-d6, ref=2.50), δ, ppm: 13.201 (1H, s, a), 9.137(1H, dd, 1), 8.611 (1H, dd, j), 8.438 (1H, s, b), 8.231 (1H, d, h),8.219 (1H, s, p), 7.955 (1H, d, i), 7.880 (1H, dd, k), 7.671 (1H, dd,n), 7.062 (1H, dd, o).

Example 18b Synthesis of P(TP)8DPCA-20K

Eight arm polyethylene glycol (P(TP)8OH-20K) (1 g, 0.369 mmol OH, 20kDa, tripentaerythritol core, JenKem) was dissolved in 4 mL DMF, using aheat gun to help dissolve the polymer. The reaction vessel was purgedwith argon, and NaH was added (8.85 mg, 0.369 mmol), with stirring underargon at room temperature for ˜30 minutes until effervescence ceased.DPCA-I (107 mg, 0.369 mmol) was added and stirred under argon at roomtemperature for 18 hours. PEG-(NH₂)₂-2K (369 mg, 0.369 mmol, 2 kDa,JenKem) was added and stirred for 30 minutes. The product wasprecipitated with 40 mL cold Et₂O, spun (4500 RCF, −5° C., 5 min),followed by decanting of the supernatant. Residual ether was removedwith N₂, and the product was redissoved in 20 mL MeOH. After cooling at−20° C. for 30 minutes, the precipitate was spun down (4500 RCF, −5° C.,5 min), and the supernatant decanted. Methanol precipitation wasrepeated twice using 40 mL MeOH each time, with cooling at −20° C. for45 minutes instead of 30. The precipitate was redissoved byhand-warming, then reprecipitated with 60 mL cold ether. The product wasspun down and the supernatant was decanted. The product was washed with30 mL ether and dried on high vacuum to yield 992 mg (91.3%)P(TP)8DPCA-20K with 38% end group activation (i.e. 3 of the 8 OH groupswere activated with DPCA). ¹H NMR (500 MHz, DMSO-d6, ref=2.50), δ, ppm:9.110 (3H, dd, 1), 8.583 (3H, dd, j), 8.573 (3H, s, b), 8.245 (3H, d,h), 7.909 (3H, d, i), 7.854 (3H, dd, k), 4.327 (6H, t, t), 3.512 (1969H,m, q+r+s).

Example 18c Drug Release from PEG

A solution of 1% w/v P(TP)8DPCA-20K was prepared in pH 7.4 buffer and 1mL aliquots were made. The polymer solution was incubated at 37° C., andat various time points, an aliquot was lyophilized. The dried materialwas redissolved in DMSO and NMR was used to quantify ester hydrolysis(spectra not shown).

Example 19

While Examples 11 and 18 illustrate several drug conjugates inaccordance with certain non-limiting embodiments of this invention,various other conjugates are contemplated, including those compoundscomprising 1,4-DPCA coupled to any of the polyol-poly(alkylene oxide)macromolecules described in the aforementioned incorporated, co-pending'744 application. Likewise, various other compounds or compositionscomprising other therapeutic agents of the sort described herein can beprepared or formulated using one or more such macromolecules, hydrogelsand/or hydrogel precursor components.

While this invention has been described in conjunction with variousmacromolecules, hydrogels and/or hydrogel precursor components, itshould be understood that these descriptions are provided only by way ofexample and are not intended to limit, in any way, the scope of thisinvention. For instance, without limitation, the compounds,compositions, methods and/or delivery systems of the present inventioncan be considered in the context of various other macromolecules,hydrogels and/or hydrogel precursors comprising a range of alkyleneoxide polymer and/or copolymer components, such macromolecules,hydrogels and hydrogel precursors as are described in the aforementionedincorporated co-pending '744 application or as would otherwise beunderstood by those skilled in the art as commercially or otherwiseavailable using synthetic techniques of the sort described therein orstraight-forward modifications thereof.

Throughout this specification, the words “comprise”, “comprises”, and“comprising” are to be interpreted inclusively rather than exclusively.The words “consist”, “consisting”, and its variants, are to beinterpreted exclusively, rather than inclusively. It should beunderstood that while various embodiments in the specification arepresented using “comprising” language, under various circumstances, arelated embodiment is also be described using “consisting of” or“consisting essentially of” language. It is to be noted that the term“a” or “an”, refers to one or more, for example, “a drug,” is understoodto represent one or more drugs. As such, the terms “a” (or “an”), “oneor more,” and “at least one” are used interchangeably herein.

As used herein, the term “about” means a variability of 10% from thereference given, unless otherwise specified.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs and byreference to published texts, which provide one skilled in the art witha general guide to many of the terms used in the present application.

We claim:
 1. A drug delivery system for epimorphic regeneration, saidsystem comprising a first macromonomer component of a formula

a second macromonomer component of a formula

wherein Cys is an N-terminal cysteine residue, each of R₁ and R₂ isindependently selected from hexaglycolic and tripentaerythritolicmoieties, each of n1 and n2 is an integer independently selected from 1to about 201; and a drug component for up-regulation of hypoxiainducible factor 1a (H1F1a), said drug component selected from prolinehydroxylase inhibitor compounds and prodrugs thereof, a said prodrugcomprising a said proline hydroxylase inhibitor compound coupled to acleavable polymer component, said drug component in an amount to producea regenerative response.
 2. The drug delivery system of claim 1 whereineach said macromonomer component is in a fluid medium.
 3. The drugdelivery system of claim 2 wherein said first and second macromonomercomponents are cross-linked to provide a hydrogel, and said drugcomponent is dispersed therein.
 4. The drug delivery system of claim 1wherein a said drug component is selected from1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA), apoly(alkylene oxide) coupled prodrug of 1,4-DPCA, dimethyloxallylglycine (DMOG),30-amino-3,14,25-trihydroxy-3,9,14,20,25-pentaazatriacontane-2,10,13,21,24-pentone(DFX), 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine (Imiquimod)and CoCl₂.
 5. The drug delivery system of claim 4 wherein a said drugcomponent is a poly(alkylene oxide) prodrug of 1,4-DPCA.
 6. A drugdelivery system for epimorphic regeneration, said system comprising afirst macromonomer component of a formula

a second macromonomer component of a formula

wherein Cys is an N-terminal cysteine residue, each of R₁ and R₂ isindependently selected from hexaglycolic and tripentaerythritolicmoieties, each of n1 and n2 is an integer independently selected from 1to about 201; and a drug component for up-regulation of hypoxiainducible factor 1a (H1F1a), said drug component selected from prolinehydroxylase inhibitor compounds and prodrugs thereof, a said prodrugcomprising a said proline hydroxylase inhibitor compound coupled to acleavable poly(alkene oxide) block copolymer component, said drugcomponent in an amount to provide an H1F1a level for induction of aregenerative response.
 7. The drug delivery system of claim 6 whereinsaid first and second macromonomer components are cross-linked toprovide a hydrogel, and said drug component is dispersed therein.
 8. Thedrug delivery system of claim 6 wherein a said drug component isselected from 1,4-DPCA, a poly(alkylene oxide) coupled prodrug of1,4-DPCA, DMOG, DFX, Imiquimod and CoCl₂.